DESCRIPTION: Alu elements are retroposons specific to the primate genome that arose from the pol III transcribed 7SL gene. There are hundreds of thousands of copies in the genome; in fact, the element makes up about 10 % of the human genome. Retrotransposition by definition increases copy number, and Alu elements are thus very successful retroposons. The long term goal of this research is to establish a system to study the retrotransposition of Alu elements. In the prior grant period, the investigator and others have demonstrated that specific Alu transcripts are rare, and Alu expression is apparently down-regulated at many levels. To follow up on these findings, first Specific Aim of the current proposal is to use transfection studies to identify the key regulatory determinants of Alu expression. Templates for SA1 include isolated Alu source gene constructs and chimeric constructs from the 7SL RNA gene enhancer, a natural pol III terminator, and Alus representing different subfamilies. 1A) These constructs will be compared to determine the effects of 5' flanking sequences, RNA structure, efficient termination and transcribed 3' flanking sequence on the steady state abundance of Alu RNA. 1B) Nuclear run-on assays will distinguish between transcriptional activity and RNA stability in affecting steady state abundance. 1C) Processing, subcellular fate, and activity of marked templates will be determined using S1, primer extension, and Northern blot assays. Experiments in SA1 will emphasize the mechanisms by which heat shock and other cellular stressors stimulate Alu transcription. Another major finding of the prior funding period is that Alu repeats exhibit different patterns of methylation in somatic tissues and in male and female germ line tissues. In males, a subset of Alus containing a BstUI site (CGCG) is hypomethylated relative both to methylation in somatic tissues and female germ line. This is in contrast to the hypermethylation pattern of genes and L1 elements in the male germ line. This study demonstrated that 75% of the PV subfamily is unmethylated in sperm, but these are almost completely methylated in spleen. Despite the undermethylation of this subset of Alus, the majority of Alus are methylated in sperm. This subset of PV Alu elements may be acting as a marker to identify the genetic contribution of the male and female. The second Specific Aim of the proposed work is to examine the possible role of Alu methylation in genomic imprinting. Based on the results in SA1, transgenic mice will be constructed with human Alu elements. 2A) The methylation state of endogenous B1 and B2 repeats and of the transgenic human Alus will be determined in various tissues including the developing male and female germ lines. Alu methylation will also be determined in preimplantation monkey embryos. 2B) The methylation state of B1 and B2 repeats mapping near imprinted genes will be determined in embryonic tissues and correlated with neighboring gene expression. 2C) The imprinted inheritance of methylation in Alu transgenes and endogenous B1 and B2 elements will be determined in F1 hybrid embryos resulting from crosses between mouse species and between Alu transgenic and nontransgenics. A potential bonus of this SA is that the Alu transgene may undergo retrotransposition. In such a case it would be possible to follow these events in vivo in an animal that offers the advantage of a little or no endogenous Alu background.